Diminishing toxicity of antiviral nu6-(hydroxyalkyl) adenines with 4-hydroxypyrazolo(3, 4-d) pyrimidine



United States Patent DIMINISHING TOXlClTY 0F ANTIVIRAL N -(HY- DROXYALKYDADENINES WITH 4-HYDROXY- PYRAZOL0(3,4-'d)PYRIMIDINE Jack E. Gray, Richland Township, Kalamazoo County,

Gerald E. Underwood, Charleston-Township, Kalamazoo County, and Kingsley M. Mann, Portage Township, Kalamazoo County, Mich, assignors to The Upjohn Company, Kalamazoo, Mich., a corporation of Delaware No Drawing. Filed Dec. 26, 1963, Ser. No. 333,658

4 Claims. (Cl. 167-65) This invention relates to antiviral compositions containing N -(hydroxyalkyl)a-denines and 4-hydroxypyrazolo(3,4-d)pyrimidine and to a method for, their use.

The primaryingredients .employedin. the novel. compositions and method of this invention are (l) N -(hydroymethyl)adenine and N -(2hydroXyethyl)-adenine of the formula I in which R ismethylene or ethylene, and (2) 4-hydroxypyrazolo(3,4-d)pyrimidine of the formula 1L I\\N/ NH 11 and the physiologi-cally acceptable acid addition salts of N -(hydroxymethyl adenine, N (Z-hydroxyethyl) adenine and 4-hydr0xypyrazolo(3,4-d)pyrimidine, such as the hydrochlorides, sulfates, phosphates, citrates, succinates, maleates, tartrates and the like. Also specifically included are the tautomers and polymorphs of the compounds of Formulas I and II. The method hereof is practiced by administering to living subjects, substantially simultaneously, an N -(hydroxyalkyDadenine and 4-hydroxypyrazolo(3,4-d)pyrimidine or their physiologically acceptable acid addition salts, all as defined above.

The N -(hydroxyalkyl)adenines can be prepared as described in US. Patent No. 2,844,577 and 4-hydroxypyrazolo(3,4-d) pyrimidine prepared as described by Robins, J. Am. Chem. Soc. 78:784 (1956).

The 'N -(hydroxyalkyl)adenines have been found effective in reducing virus titers in living subjects. For example, in mice infected with Coe virus the paralytic aspects of the disease have been notably modified, the number of deaths reduced and virus titers in certain organs and tissues significantly lowered by such antiviral treatment. Significant reduction in titers has also been shown against PR-8 virus. However, the usefulness of the N 1(hydroxyalkyl)adenines is circumscribed by toxicity at the higher doses sometimes employed. Unexpectedly, combined therapy comprising N -(hydroxymethyl)adenine or N -(2-hydr0xyethyl)adenine with 4- hydroxypyrazolo(3,4-d) pyrimidine has been found to provide unimpaired antiviral efficacy with significantly diminished toxicity.

Pharmaceutical compositions containing the presently described ingredients, based on activity demonstrated against representative viruses, alter favorably the course of viral diseases in mammals caused by respiratory viruses such as Coe virus. Benefits from treatment of mammals and birds derive from reduction of virus titers, interference with a secondary viral effect with consequent shortening of the illness, prevention of infection by a regimen of prophylactic medication, and interference With the metabolism of the host cell to decrease virus synthesis.

The principal ingredients are conveniently incorporated in animal and bird feed carriers of both the premix and readymix types. The former contain edible diluents such as starch, oatmeal, flour, licalcium phosphate, talc, dried fish meal, soybean meal and the like non-toxic orally acceptable diluents. The prepared premix is then conveniently added'to the regular ration, thereby providing medication to the bird or animal in the course of its consumption of the said ration. The ready-mixed type is prepared by incorporating the principal ingredients directly into the ration for consumption without further dilution. The animal and bird'feed preparations can be of both the solid and liquid types.

In addition to the N -(hydroxyalkyl)adenines and 4- hydroxypyrazolo( 3,4-d) pyrimidine, other complementary ingredients can be included in the compositions to secure advantageous combinations of properties especially adapted to individual situations in the treatment of the foregoing conditions. In general, the amounts of such complementary ingredients employed in each unit dosage do not exceed the individual unit doses normally employed when such ingredients are administered alone. Thus, combinations of the primary active ingredients with analgesics such as aspirin (200-400 mg), N-acetyl-paminophenol (200-400 mg.) and salicylamide (200-400 mg); antihistamines such as chlorpheniramine maleate (2-4 mg, pyrathiazine hydrochloride (10-50 mg), promethazine hydrochloride (5-30 mg.) and pyrilamine maleate (15-60 mg); decongestants such as hydroxyamphetamine hydrobromide (0.5-1% phenylephrine hydrochloride (0.1-0.5 phenylpropanolamine hydrochloride (0.5-1.5 antibitoics such as penicillin (l00,- GOO-1,000,000 U.), tetracycline (l-3% topically; 50-500 mg. systematically); neomycin (0.1-2%), bacitracin (200-500 U./cc.) other antivirals such as l-fl-D-arabinofuranosylcytosine (0.52.5% topically; 50-500 mg. systemically) N',N'-anhydro (bis) p-hydroxyethyl:biguanide hydrochloride (05-25%), and 5iodo-2'-deoxyuridine (05-25% and other coacting materials such as vitamin C (-500 mg.) can be employed.

Suitable oral dosage forms include tablets, pills, capsules, granules, powders, oral solutions or suspensions and the like. For preparing solid compositions such as tablets, the active ingredient is admixed wi-th conventional tableting ingredients or pharmaceutical carriers such as cornstarch, lactose, dioalcium phosphate, terra alba (calcium sulfate), talc, stearic acid, magnesimn stearate, gums and functionally similar materials serving as lubricating agents, granulating agents, pharmaceutical diluents or carriers. The tablets or pills of these novel compositions can be laminated or otherwise compounded to provide a dosage form affording the advantages of prolonged, delayed or predetermined successive action of the enclosed medication. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope enclosing the former. The two components can be separated by an enteric layer which serves to resist disintegration until the desired site of release is encountered. A variety of materials can be used for such enteric layers or coatings, these materials including a number of polymeric acids or mixtures of polymeric acids with, for example, shellac, shellac and cetyl alcohol, cellulose acetate phthalate, and the like. Advantageously, a coating consisting essentially or gelatin and styrene-maleic acid copolymer, deposited on the drutb particles by a process of phase separation, such as by coacervation, can be employed as the encapsulating material capable of affording sustained release of the enclosed medication.

The oral liquid forms in which the novel compositions of this invention can be incorporated include aqueous Therefore, the score for a given group on a given day represented one-half the sum of the scores of the 20 individual mice in that group. This score was compared to the average obtained on the infected controls. Statissolutions, flavored syrups, elixirs, suspensions, flavored 5 tical analysis indicated that for significant activity, with emulsions with edible oils such as cottonseed oil, sesame 95% confidence limits, the score for the treated group oil, coconut oil, peanut oil and the like. Suitable dispersshould exceed the control score by 15 units on the 6th ing or suspending agents for aqueous suspensions include day reading or by 20 units on the 10th day reading. synthetic and natural gums such as tragacanth, acacia, Results were as follows: alginates, dextran, methyl-cellulose, polyvinylpyrrolidone, 10 gelatin and the like. For veterinary use an aqueous sus- 6 Day Ito-Day pension prepared as above and sterilized offers a con- Treatment fig venient form for administration by injection. y Score A Score A Topical compositions incorporating the primary ingredients hereof are those prepared in such conventional carboxymethylgellulose, pharmaceutical forms as eye, ear, throat and nasal sprays, gzgf g d fdig fi fis: 32 4 drops, powders for inhalation and the like. adenine (A) 50 59 27 31 21 The term unit dosage form as applied to the composigg t p-"f pyrimidine (B) -0 3.; 3 5 1 t1ons of this inventlon refers to physically d1screte units (A)+(B) 50+20 40 14 36 32 suitable as unitary dosages, each unit containing a predetermined quantity of active material intended to pro- A=Score of treated minus score ofcontrols. duce.the dlelslred f igl effect m a ii 1 2? The foregoing results show N -(2-hydroxyethyl)adearmacegtlca i gamer 1c 6 nine to be active, 4-hydroxypyrazolo(3,4-d)pyrimidineto spec? f govel i g Is mbe inactive, and the combination of the two to exhibit $5 $3 fii chiia iferaia fif thi iai ve .532521 5511 iii activity Comparable to Nsieh-vdroxyehynadenine articuliir thera eutic effect to be achieved and (b) the In View of the a'Rpa-rent absence of eff-sq of 4'h-yc-lroxy' l imitations inhe ent in the art of compounding such an pyrazolo(34 d)pynmldin? on the anilvlral actmty of active material for therapeutic use as disclosed in de- Ns'(.zhydroxyeth.yl)adenme at non'tofuc levels of the tail herein these bein features of the present invention It was then Important to determine the efiect of 4 D hydroxypyrazol o(3,4-d)pyrimidine with higher doses of Examples of suitable unit dosage forms 1n accord with drox 6th 1) adenine this invention are tablets, capsules, pills, powder packets, y y y wafers, cachets, teaspoonfuls, tablespoonfuls, dropperfuls, Example 2 Segregated mlfltip'les of of the foregoing and other Groups comprising 10 mice each were infected as in forms as herefnbefore descn'bed- 35 Example 1 and treated twice daily for 5 days. N -(2- The followmg exjlmples 'f h best mode hydroxyethyl)adenine was injected at levels of 400, 200 templated for carry ng out th1s invention but are not to and 100 bodyweight per day 4 hydroxypyrazolo be construed as hmltmg the scope thereof- (3,4-d)pyrimidine was given at a constant level of 20 mg./kg. bodyweight per day, and combinations of N Example 1 40 (2-hydroxyethyl)adenine and 4-hydroxypyrazolo(3,4-d) Four groups of 20 mice each were infected intracerepyrimidine were administered daily as indicated. Deaths brally with Coe Stock 8 virus (0.03 ml. of a 10% muscle and average weights of survivors were recorded for each suspension harvested from infected mice at the time they group on the 1st, 4th, 7th and 11th days. Results were showed marked paralysis). Treatment was by the intraas follows:

Day 1 Day 4 Day 7 Day 11 Treatment" Dose,

mgJkg/day Wt. gm. i Dead/Total AWt. Dead/Total AWt Dead/Total AWt. Dead/Total *A N (Z-hydroxyethyl) adenine. B 4-hydroxypyrazolo (3,4-(1) pyrimidine.

peritoneal route, starting one day before infection and continuing twice daily for five successive days. Compounds were prepared in aqueous 1% sodium carhoxymethylcellulose solution. The first group received N (Z-hydroxyethyDadenine only, mg./kg. bodyweight per day. The second group received 4-hydroxypyrazolo- (3,4 d)pyrimidine only, 20 mgJkg. bodyweight per day. The third group comprised untreated control animals which were given comparable amounts of 1% sodium car'boxymethylcellulose solution. Eflicacy was determined by scoring the mice by reference to a graded scale ranging from apparent good health, through varying degrees of paralysis, to death. Each mouse was examined on the 6th and 10th day after inoculation. Grading was as follows: normal appearance, 10; slight paralysis, 7; paralyzed, 5; extreme paralysis, 2; dead, 0. For convenience, it was desired that a total score of 100 represent complete protection for each test group of 20 mice.

The foregoing results show that 4-hydroxypyrazolo- (3,4-d)pyrimidine, when administered in conjunction with N -(2-hydroxyethyl)adenine markedly decreases the toxic effects of the antiviral agent.

Example 3 .Tablets One thousand tablets for oral use, each containing 250 mg. of N -(2-hydroxyethyl)adenine and 25 mg. of 4- hydroxypyrazolo(3,4-d)pyrimidine, are prepared from the following ingredients:

The powdered primary ingredients are granulated with a 4% W./v. aqueous solution of methylcellulose, 1500 .5 cps, U.S.P. To the dried granules is added a'mixture of the remainder of the ingredients. The final mixture is'compressed into tablets of proper weight.

Example 4.-Har i gelatin capsules One thousand two-piece hard gelatin Capsules for oral use, each containing 1000 mg:-of Nfi-(hydroxyrnethyl) adenine and 40 mg. of 4-hydroxypyrazolo(3,4-d)pyrimidine, are prepared from the following ingredients:

N -(hydroxymethyl)adenine 1000 4-hydroxypyrazolo 3 ,4-d pyrimidine 40 Corn starch, U.S.P. 1875 Light mineral oil, U.S.P. 130 Magnesium stearate powder 160 Talc, U.S.P. 160

The materials are mixed thoroughly and encapsulated.

Example 5.-Sft elastic capsules One thousand soft-elastic capsules-for oral use, each containing 50 mg. of N -(2-hydroxyethyl)adenine hydrochloride and 50 mg. of 4-hydroxypyrazolo(3,4-d)pyrimidine hydrochloride, are prepared by dispersing 50 gm. each of said ingredients in sufiicie-nt corn oil to render the drugs capsulatab le and then encapsulating in the usual manner.

Example 6.*-Aque0us preparation Ten liters of aqueous preparation for oral use, containing in each tablespoonfuil (15 ml.) 1500 mg. of N -(hydroxymethyDadenine hydrochloride and 500 mg. of 4-hydroxypyrazolo(3,4-d)pyrimidine hydrochloride, are prepared by thoroughly mixing together the following materials:

Example 7.Veterinary pre-mix Ten thousand grams of a pre-rnix is prepared by thoroughly mixing 1000 gm. of N -(2-hydroxyethyl)adenine hydrochloride and 500 gm. of 4-hydroxypyraZolo(3,4-d) pyrimidine hydrochloride into 8500 gm. of soybean meal. Each gram of the pre-mix thus contains 100 mg. of the adenine and 50 mg. of the pyrazolopyrimidine. The premix is added to the standard ration of cattle to provide a daily dose of 10 gm. of the adenine.

Example 8.-Veterinary bolus Ten thousand boluses, each containing gm. of N -(2- hydroxyethyl)adenine and 2.5 gm. of 4-hydroxypyrazolo (3,4-d)pyrimidine, are prepared from the following ingredients:

N -(2-hydroxyethy l) adenine 50,000 4-hydroxypyrazolo 3,4-d pyrimidine 25,000

Lactose 250,000

The ingredients are blended and granulated with syrupstarch paste, and q.s. mineral oil is added. The granulation is then dried, lu bricated with starch, talc and calcium stearate powders, and compressed with a 1 /2 x die.

Example 9.Injectable preparation A sterile aqueous preparation suitable for intramuscular injection and containing 250 mg. of N -(l-hydroxyethyl) adenine and 25 mg. of 4-hydroxypyrazolo(3,4-d)pyrimidine in each 2 ml. is prepared from the following ingredients:

N 2-hydroxyethy1 adenine gm 4-hydroxypyrazolo 3,4-d) pyrimidine gm 12.5

Polyethylene glycol 4000, U.S.P.' gm 30 Sodium chloride, U.S.P. gm 9 Preservative, q.s.

Water for injection, q.s. ml- 1000 Example 10 For the principal ingredients of Examples 3 through 9 can be substituted the physiologically acceptable acid addition salts, such as the hydrochlorides, sulfates, phosphates, citrates, succinates, maleates, tartrates and the like.

What is claimed is:

1. A pharmaceutical preparation for reducing toxicity and maintaining antiviral action of a member selected from the group consisting of N -(hydroxymethyl)adenine, N -(2-hydroxyethyl)adenine and the physiologically acceptable acid addition salts thereof which comprises (1) an effective amount of said member for reducing virus titers and (2) a member selected from the group consisting of 4-hydroxypyrazolo-(3,4-d)pyrimidine and the physiologically acceptable acid addition salts thereof, wherein the ratio of the former member to the latter member is from about 2.5 :1 to about 20:1.

2. A pharmaceutical preparation for reducing toxicity and maintaining antiviral action of a member selected from the group consisting of N -(hydroxymethyl)adenine, N -(2-hydroxyethyl)adenine and the physiologically acceptable acid addition salts thereof which comprises (1) an amount of said member to provide from about 50 mtg/kg. to about 400 mg/kg. per day of said member and (2) a member selected from the group consisting of 4-hydroxypyrazolo-(3,4-d) pyrimidine and the physiologically acceptable acid addition salts thereof wherein the ratio of the former member to the latter member is from about 2.5:1 to about 20:1.

3. A method for reducing toxicity and maintaining antiviral action of a member selected from the group consisting of N -(hydroxymethyl)adenine, N -(2-hydroxyethyl)-adenine and the physiologically acceptable acid addition salts thereof which comprises the parenteral administration of a pharmaceutical preparation according to claim 1.

4. A method for reducing toxicity and maintaining antiviral action of a member selected from the group consisting of N -(hydroxymethyl)adenine, N -(2-hydroxyethyDadenine and the physiologically acceptable acid addition salts thereof which comprises the parenteral adminstration of a pharmaceutical preparation according to claim 2.

References Cited by the Examiner UNITED STATES PATENTS 3/1966 Underwood 167-53 FOREIGN PATENTS 7/1958 Great Britain.

(Other references on following page) pages 1483 to 1491, 1493 to 1494, 1498, 1636 (entry 5 41723), September 1963.

8 Pomales et a1.: Biochem. Biophys. Acta 72(1): 119 to 120, May 28, 1963.

Stock et a1.: Cancer Research 18 (N0. 8, part 2), pages 49 to 53, 102: (entry 7515) September 1958.

LEWIS GO'ITS, Primary Examiner.

SHEP K. ROSE, Assistant Examiner. 

1. A PHARMACEUTICAL PREPARATION FOR REDUCING TOXICITY AND MAINTAINING ANTIVIRAL ACTION OF A MEMBER SELECTED FROM THE GROUP CONSISTING OF N6-(HYDROXYMETHYL) ADENINE, N6-(2-HYDROXYETHYL)ADENINE AND THE PHYSIOLOGICALLY ACCEPTABLE ACID ADDITION SALTS THEREOF WHICH COMPRISES (1) AN EFFECTIVE AMOUNT OF SAID MEMBER FOR REDUCING VIRUS TITERS AND (2) A MEMBER SELECTED FROM THE GROUP CONSISTING OF 4-HYDROXYPYRAZOLO-(3,4-D)PYRIMIDINE AND THE PHYSIOLOGICALLY ACCEPTABLE ACID ADDITION SALTS THEREOF, WHEREIN THE RATIO OF THE FORMER MEMBER TO THE LATTER MEMBER IS FROM ABOUT 2.5:1 TO ABOUT 20:1. 